Dengue serotype 1 vaccine development: The live attenuated DEN1 vaccine candidate virus rDEN1del30 has been evaluated in Phase I clinical trials and was found to be safe and immunogenic at a dose of 1000 PFU. An additional study has been completed to determine the number of doses and the timing of the two doses needed to achieve optimal immunization. The study involved 60 healthy flavivirus-naive adults randomized to receive 2 doses of rDEN1del30 (N=50) or placebo (N=10), either on study days 0 and 120 (cohort 1) or 0 and 180 (cohort 2). We sought to evaluate the safety and immunogenicity of this candidate vaccine in 50 additional vaccinees and to test whether the humoral immune response could be boosted by a second dose administered 4 or 6 months after the first dose. The first dose of vaccine was well tolerated, infected 47/50 vaccinees and induced seroconversion in 46/50 vaccinees. Irrespective of dosing interval, the second dose of vaccine was also well tolerated but did not induce any detectable viremia or &#8805;4-fold rise in serum neutralizing antibody titer. Only five subjects had an anamnestic antibody response detectable by ELISA following a second dose of vaccine, demonstrating that the vaccine induced sterilizing humoral immunity in most vaccinees for at least six months following primary vaccination. A separate clinical trial to determine the 50% infectious dose (HID50) was recently completed and showed that rDEN1del30 is highly infectious, even at the lowest dose administered (10 PFU). Dengue serotype 2 vaccine development: rDEN2/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine, rDEN4del30, have been replaced by those of the prototypic DEN2 NGC virus. The live attenuated DEN2 vaccine candidate virus rDEN2/4del30(ME) has been evaluated in Phase I clinical trials and found to be safe and immunogenic at a dose of 1000 PFU. Additional studies have been completed to address the number and timing of two doses of this vaccine candidate. Results obtained this year are similar to those observed previously with rDEN1del30 and showed that a second dose administered at 6 months did not boost the immune response to the primary dose, presumably due to complete neutralization of the second dose by the existing antibody. A separate clinical trial to determine the 50% infectious dose (HID50) was recently completed and showed that at the lowest dose administered (10 PFU), approximately half of the volunteers seroconverted to DEN2, indicating that the HID50 is close to 10 PFU. Dengue serotype 3 vaccine development: Due to the observed low infectivity of chimeric vaccine candidate rDEN3/4del30(ME), two additional DEN3 vaccine candidates were studied in which 1) the 3 UTR (untranslated region) of rDEN3 was replaced with that derived from attenuated rDEN4del30 to generate rDEN3-3D4del30, and 2) a pair of 3 UTR deletions were introduced into DEN3 to create vaccine candidate rDEN3del30/31. Clinical evaluation showed that the rDEN3-3D4del30 vaccine was well-tolerated and 80% of the volunteers were infected. In a separate clinical trial, the rDEN3del30/31 vaccine was shown to be well-tolerated with 95% of the volunteers becoming infected. Both of these new DEN3 vaccine candidates were evaluated in tetravalent vaccine formulations. Dengue serotype 4 vaccine development: We currently consider the rDEN4del30 vaccine our leading DEN4 candidate for inclusion in the tetravalent vaccine. However, rDEN4del30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4del30 parent virus. In a recent clinical study, the vaccine infected 95% of vaccinees and was well tolerated without inducing ALT elevations. Although virus was not recovered from the serum of any vaccinee, moderate levels of neutralizing antibody were induced. Both the rDEN4del30 and rDEN3del30/31 vaccine candidates were evaluated in tetravalent vaccine formulations. Tetravalent studies: Phase I clinical studies were completed this year to evaluate four different tetravalent formulations containing 1000 PFU of each vaccine candidate. Each formulation contained rDEN1del30 and rDEN2/4del30 along with the following DEN3 and DEN4 vaccine candidates: TV001 contained rDEN3-3D4del30 and rDEN4del30, TV002 contained rDEN3-3D4del30 and rDEN4del30-200,201, TV003 contained rDEN3del30/31 and rDEN4del30, and TV004 contained rDEN3del30/31 and rDEN3del30/31. Each formulation was evaluated as a single dose in 20 vaccinees and 8 placebo recipients. Preliminary results indicate that among the different formulations, the percentage of seroconversion to DENV serotypes 1, 2, 3, and 4 were 80 100%, 50 65%, 60 85%, and 85 100%, respectively, with TV003 achieving the highest percent seroconversion (100, 50, 85, and 100% to the four serotypes). Among the recipients of TV-003, 90% seroconverted to at least 3 serotypes (40% seroconverted to all 4 serotypes) after a single inoculation. A fifth formulation is currently undergoing clinical evaluation and contains a 10-fold higher dose of the DENV-2 component in an attempt to increase seroconversion to DENV-2. Heterotypic dengue vaccination: Since infection with any of the four DENV serotypes may elicit both protective neutralizing antibodies and non-neutralizing antibodies capable of enhancing subsequent heterotypic DENV infections, the greatest risk for severe dengue occurs during a second, heterotypic DENV infection. It remains unclear whether the replication of live-attenuated vaccine viruses will be similarly enhanced when administered to DEN-immune individuals. We recruited 36 healthy adult subjects who had previously received a monovalent live DENV vaccine 0.6 7.4 years earlier. Subjects were assigned to one of four cohorts and randomly chosen to receive placebo or a heterotypic vaccine. The level of replication, safety, and immunogenicity of the heterotypic vaccine virus was compared to that of DENV immunologically naive vaccinees. Results showed that vaccine virus replication and the reactogenicity following monovalent DENV vaccination in naive and heterotypically immune vaccinees was similar. In contrast to naive vaccinees, the antibody response in heterotypically immune vaccinees was broadly neutralizing and mimicked the response observed by natural secondary DENV infection. Overall, the enhancement of replication of these live-attenuated DENV vaccines was minimal in heterotypically-immune vaccinees and supports the further evaluation of these candidate vaccines in populations with pre-existing DENV immunity.